Primary hepatocellular carcinoma is carcinoma with poor prognosis, which ranked third (13%) in men and fourth (9.0%) in women with regard to death by carcinoma, a primary cause of death in Japan in 2001 (excerpt from “Population Survey Report” Statistics and Information Department, Minister's Secretariat, the Ministry of Health, Labour, and Welfare). The number of chronic patients has increased due to virus infection year after year, and a majority of such patients develop hepatocirrhosis and then hepatocellular carcinoma in many cases. Under such circumstances, it has been extremely strongly desired that an early diagnostic method applied at stages ranging from such hepatocirrhosis to hepatocellular carcinoma and a method for treating hepatocellular carcinoma be developed. If a breakthrough resolution were not found, the number of people dying would increase over the next 10 to 15 years. As a diagnostic method, such hepatocellular carcinoma is comprehensively evaluated on the basis of biochemical data such as the values of GOP/GTP, alkaline phosphatase, albumin, etc. in serum or the value of AFP (α-photoprotein) used as a tumor marker, and diagnostic imaging. Thereafter, if necessary, a small amount of piece of tissue is collected by a needle biopsy, and a confirmed diagnosis is carried out based on pathological determination. At present, a tumor marker has been used particularly in the diagnosis of hepatocellular carcinoma The positive rate of α-photoprotein (AFP), the most commonly used tumor marker, in patients with hepatocellular carcinoma is approximately 60 to 70%. However, such a positive result may also be obtained, when a subject is a patient with a chronic liver disease or a pregnant woman. In addition, the positive rate of PIVKA-II, a tumor marker for hepatic carcinoma, is low (somewhat lower than 50%), but the specificity of this marker to hepatocellular carcinoma is considered higher than that of AFP. Currently, such two types of examinations have mainly been conducted. At any rate, since false positive or double negative cases exist, it is anticipated that a tumor marker having high specificity be developed.
In recent years, a gene analysis technique using a high-performance array such as a DNA microarray has been developed, and thus all-inclusive and comprehensive analyses of gene expression in carcinoma have become practicable. A change in the expression level of mRNA in cancer tissues is analyzed by a DNA microarray analysis method, and a gene group associated with malignant degeneration of carcinoma due to multistep factors, invasion or metastasis of cancer cells, etc. has been comprehensively identified. Moreover, it is expected that several numbers of new findings regarding the new properties of cancer cells will be obtained by clarifying the individual physiological functions of such an identified gene group. Thus, identification of molecules, the expression of which is accentuated or decreased in various types of carcinomas, has been progressing.
ROBO1, a hepatocellular carcinoma-specific expression molecule, is a type I membrane protein that is a member of the immunoglobulin superfamily, to which N-CAM, DCC, L1-CAM, etc. belong. ROBO1 has 5 immunoglobulin domains and 3 fibronectin III domains in the extracellular region thereof. The amino acid sequence is highly conserved in various organisms ranging from a fly to a human. It shows amino acid sequence homology of 34% with C. elegans, 33% with Dorosofila, 96% with a mouse, and 95% with a rat. A fly homolog of ROBO1 has been cloned as a molecule that controls the median crossing of an axial filament in gene screening studies of fruit flies. It has been reported that such a fly homolog is a Slit protein receptor. In addition, by another study group, ROBO1 has also been identified as Dutt1 (Deleted U Twenty Twenty), a molecule existing in a homozygous missing region in the chromosomal region 3p12 of U2020 that is a cell line of lung small cell carcinoma With regard to ROBO1, loss of heterozygosity (LOH) at chromosome is detected at a high frequency in lung cancer, breast cancer, and kidney cancer. Due to methylation of a promoter region in the other allele, the expression thereof is suppressed. Such facts suggest the possibility of ROBO1 as a cancer suppressor gene. In the case of ROBO1 homozygous deficient mice, it has been reported that half of the mice die during the prenatal period, and that the surviving half mice also lead to death due to pulmonary hypoplasia. In addition, in the case of ROBO1 heterozygous deficient mice, the incidence of cancer 1 year or more after the birth is 3 times higher than that of normal mice. From this fact as well, ROBO1 is considered to be a cancer suppressor gene. Moreover, the expression of a Slit2 gene that is a ROBO1 ligand is also suppressed by methylation or the like in many types of carcinomas. In a test using a conditioning medium of Slit2 or the like, Slit2 exhibited proliferative inhibitory action and apoptotic action towards a lung cancer cell line, a breast cancer cell line, and a colon cancer cell line. Thus, Slit2 is also considered to be a candidate molecule for a cancer suppressor gene. As a completely different finding, Wang et al. has reported that ROBO1 is expressed in neovascularity, and that the expression of Slit2 is accentuated in cancer cells and it is associated with vascularization in cancer cells.
Furthermore, identification of ROBO1 as a hepatocellular carcinoma antigen, the potential treatment of hepatocellular carcinoma using an anti-ROBO1 monoclonal antibody towards a ROBO1 antigen as a target, and the potential serodiagnosis of hepatocellular carcinoma using a soluble-type ROBO1 as an indicator, have also been reported (Non-patent document 1).
On the other hand, the diagnostic imaging of tumor is carried out using CT or MRI. However, in some cases, it is difficult for CT or MRI to carry out differentiation of benign tumor from malignant tumor, diagnosis of recurrence after surgery, differentiation of the tumor from other pathologic lesions, even using a contrast medium. Recently, Positrom Emission Tomography (PET) using 18F-2-fluoro-2-deoxyglucose (18F-FDG) has been used as a diagnostic imaging to assist CT or MRI. However, since 18F-FDG is accumulated even in normal tissues (for example, brain, etc.) involving active glucose metabolism or in inflammatory tissue at an acute stage, as a diagnostic mechanism using 18F-FDG, it has been difficult to make a diagnosis in some types of a tumor. Moreover, in drug discovery, molecular imaging is a technique of visualizing a small amount of molecule existing in a living body, such as a protein molecule in a living body, using such amount as an indicator, and further quantifying the dynamics. In particular, such a method using PET is excellent in terms of detection sensitivity and quantitative capability. In particular, PET used for small animals is expected to drastically accelerate in-vivo translational research and to have an impact on innovative changes in a drug discovery process.
Non-patent document 1: Ito H, et al, Identification of ROBO1 as a nobel hepatocellular carcinoma antigen and a potential therapeutic and diagnostic target. Clin Cancer Res. 2006 Jun. 1; 12(11 Pt 1):3257-64.